Sonosensitive liposome, pharmaceutical composition including the same, and method of delivering active agent to subject using the sonosensitive liposome

ABSTRACT

Provided is a liposome comprising a lipid bilayer and a sonosensitizer that is disposed in and/or on the lipid bilayer, wherein the sonosensitizer self-assembles to form aggregates when exposed to ultrasound; and a method of efficiently delivering an active agent to a target site in the body of a subject using the sonosensitive liposome.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of Korean Patent Application No.10-2013-0147520, filed on Nov. 29, 2013, in the Korean IntellectualProperty Office, the entire disclosure of which is hereby incorporatedby reference.

BACKGROUND

1. Field

The present disclosure relates to sonosensitive liposomes,pharmaceutical compositions including the same, and methods ofefficiently delivering an active agent to a subject using thesonosensitive liposomes.

2. Description of the Related Art

Liposomes have at least one lipid bilayer membrane enclosing an aqueousinternal compartment. Liposomes may be characterized by membrane typeand by size. Small unilamellar vesicles (SUVs) have a single membraneand have a diameter in a range from about 20 nm and to about 50 nm.Large unilamellar vesicles (LUVs) may have a diameter of at least 50 nm.Oligolamellar large vesicles and multilamellar vesicles have multiple,usually concentric, membrane layers, and may have a diameter of at least100 nm. Liposomes with several nonconcentric membranes, i.e., severalsmaller vesicles contained within a larger vesicle, are termedmultivesicular vesicles.

Liposomes are formulated to carry drugs or other active agents eithercontained within an aqueous interior space (water-soluble active agents)or partitioned into the lipid bilayer (water-insoluble active agents).In addition, hydrophobic materials such as cholesterols are contained ina micelle, and the micelle may be contained in the liposome interiorspace.

Ultrasound-enhanced drug delivery is non-invasive and is carefullyconcentrated and controlled, and accordingly, such drug delivery mayhave several advantages including drugs may be penetrated to a targetsite deep in the body. Here, use of initial ultrasound in helping drugdelivery was transcutaneous.

Therefore, in order to efficiently deliver active agents such as drugs,new sonosensitive liposomes have been required.

BRIEF SUMMARY

Provided is a liposome comprising a lipid bilayer; and a sonosensitizerthat is disposed in and/or on the lipid bilayer, wherein thesonosensitizer self-assembles to form aggregates when exposed toultrasound.

Also provided is a method of delivering an active agent to a subject,the method comprising administering the liposome provided herein to asubject, and applying ultrasound to the liposome to release the activeagent.

BRIEF DESCRIPTION OF THE DRAWINGS

These and/or other aspects will become apparent and more readilyappreciated from the following description of the embodiments, taken inconjunction with the accompanying drawings in which:

FIG. 1 is a graph showing the extent of drug release, according to theamount of sonosensitizer and ultrasound exposure time used; and

FIG. 2 is a graph showing the effects of liposomes on cell viability,the liposomes containing drugs and being sonicated at variousamplitudes.

DETAILED DESCRIPTION

Reference will now be made in detail to embodiments, examples of whichare illustrated in the accompanying drawings, wherein like referencenumerals refer to like elements throughout. In this regard, the presentembodiments may have different forms and should not be construed asbeing limited to the descriptions set forth herein. Accordingly, theembodiments are merely described below, by referring to the figures, toexplain aspects of the present description. As used herein, the term“and/or” includes any and all combinations of one or more of theassociated listed items. Expressions such as “at least one of,” whenpreceding a list of elements, modify the entire list of elements and donot modify the individual elements of the list.

According to an aspect of the present disclosure, a sonosensitiveliposome includes a lipid bilayer and a sonosensitizer disposed inand/or on the lipid bilayer, wherein the sonosensitizer self-assemblesto form aggregates, when exposed to ultrasound.

The term “liposome” as used herein indicates an artificially and/ornaturally prepared vesicle formed of a lipid bilayer. A liposome may bein a form of unilamellar vesicles or multilamellar vesicles.

The liposome may be a sonosensitive liposome. The sonosensitive liposomerefers to a liposome that increases permeability thereof when exposed toultrasound. In this regard, when the sonosensitive liposome is exposedto ultrasound, an active agent that is contained in the sonosensitiveliposome may be released. In some embodiments, the liposome may benon-sensitive to temperatures. The active agent-containing liposome maynot have permeability changes of more than 10% at a temperature rangingbetween about 25° C. and about 45° C., for example, more than 8%, morethan 6%, more than 4%, more than 3%, more than 2%, more than 1%, or morethan 0.5%.

Ultrasound may be a wave with a frequency greater than an audiofrequency of about 16 Hz to about 20 kilohertz (kHz). Ultrasound may behigh intensity focused ultrasound (HIFU), high non-intensity focusedultrasound, or a combination thereof. HIFU is ultrasound involvinghigh-intensity ultrasound energies in one place to create a concentratedfocus. HIFU may be ultrasound-guided HIFU or magnetic resonance imaging(MRI)-guided HIFU. Ultrasound may have a frequency, for example, in arange from about 20 kHz to about 2.0 megahertz (MHz), about 40 kHz toabout 2.0 MHz, about 60 kHz to about 2.0 MHz, about 80 kHz to about 2.0MHz, about 100 kHz to about 2.0 MHz, about 150 kHz to about 2.0 MHz,about 200 kHz to about 2.0 MHz, about 250 kHz to about 2.0 MHz, about300 kHz to about 2.0 MHz, about 350 kHz to about 2.0 MHz, about 400 kHzto about 2.0 MHz, about 450 kHz to about 2.0 MHz, about 500 kHz to about2.0 MHz, about 550 kHz to about 2.0 MHz, about 600 kHz to about 2.0 MHz,about 650 kHz to about 2.0 MHz, about 700 kHz to about 2.0 MHz, about750 kHz to about 2.0 MHz, about 800 kHz to about 2.0 MHz, about 850 kHzto about 2.0 MHz, about 900 kHz to about 2.0 MHz, about 950 kHz to about2.0 MHz, about 1.0 MHz to about 2.0 MHz, about 1.1 MHz to about 1.9 MHz,about 1.2 MHz to about 1.8 MHz, about 1.3 MHz to about 1.7 MHz, or about1.4 MHz to about 1.6 MHz.

The term “lipid bilayer” as used herein refers to a membrane made of twolayers of lipid molecules. The lipid bilayer may have a thicknesssimilar with that of a naturally existing membrane, such as a cellmembrane, a nuclear membrane, and a virus envelop. For example, thethickness of the lipid bilayer may be 10 nm or less, for example, in arange from about 1 nm to about 9 nm, about 2 nm to about 8 nm, about 2nm to about 6 nm, about 2 nm to about 4 nm, or about 2.5 nm to about 3.5nm. The lipid bilayer is a barrier that keeps ions, proteins, and othermolecules where they are needed and prevents them from diffusing intoareas where they should not be. The “lipid molecule” for constructingthe lipid bilayer may be a molecule having a hydrophilic head andhydrophobic tails. The lipid molecule may have 14 to 50 carbon atoms.

The lipid bilayer may be, but is not limited to, a phospholipid, a lipidconjugated to polyethylene glycol (PEG), cholesterol, or any combinationthereof.

The phospholipid is a complex lipid containing phosphate ester within amolecule. Also, the phospholipid is a main component of biologicalmembranes, such as a cell membrane, endoplasmic reticulum, mitochondria,and myelin sheath around nerve fibers. The phospholipid has ahydrophilic head and two hydrophobic tails. When the phospholipids areexposed to water, they arrange themselves into a two-layered sheet(bilayer) with all of their tails pointing toward the center of thesheet. The center of this bilayer contains almost no water, and alsoexcludes molecules like sugars or salts that dissolve in water but notin oil. The phospholipid with a certain head group may alter the surfacechemistry of the bilayer. In addition, the lipid tails may affectmembrane properties, for instance by determining the phase of thebilayer. The lipid bilayer may adopt a solid gel phase state at lowertemperatures, but undergo phase transition to a fluid state at highertemperatures. The packing of lipids within the lipid bilayer may alsoaffect mechanical properties thereof, including resistance to stretchingand bending. A biological membrane may include several types of lipidsother than the phospholipids.

The phospholipids may include phosphatidic acid,phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phosphatidylinositol, phosphosphingolipid, or any combination thereof.Phosphatidylcholine (PC) may include choline as a head group andglycerophosphoric acid as a tail, wherein glycerophosphoric acid may besaturated fatty acid or unsaturated fatty acid, and have 14 to 50 carbonatoms. Examples of the PC include1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), egg PC, soy bean PC,or any combination thereof. The phospholipid may include, for example,DPPC and egg PC at a ratio ranging from about 4:1 to about 1:4, about3:1 to about 1:3, or about 2:1 to about 1:2. For example, thephospholipid may include DPPC and egg PC at a ratio of 1:1.

The lipid conjugated to polyethylene glycol (PEG) may be, for example,phosphatidylethanolamine (PE)-PEG. The PE may be saturated fatty acid,unsaturated fatty acid, a mixed acyl chain,lysophosphatidylethanolamine, or any combination thereof. The lipidconjugated to PEG may be, for example,1,2-distearoylphosphatidylethanolamine-methyl-polyethylene glycol(DSPE-PEG).

The term “cholesterol” as used herein refers to any one of steroidcompounds. The cholesterol may also include a cholesterol derivative,and examples thereof include sitosterol, ergosterol, stigmasterol,4,22-stigmastadiene-3-on, stigmasterol acetate, lanosterol,cycloartenol, or any combination thereof. The cholesterol may enhancestability of a lipid bilayer and assist to lower permeability of thelipid bilayer.

The term “primary lipid” as used herein refers to a main lipid componentof a liposome bilayer material in a liposome bilayer. Thus, for example,in a liposome bilayer in which 70% is phospholipid and 30% ischolesterol, the primary lipid is the phospholipid.

The sonosensitizer may be a material that increases the permeability ofthe liposomes when the liposomes are exposed to ultrasound. The increasein the permeability of the liposomes may be induced by thesonosensitizer, the sonosensitizer being self-assembled to formaggregates when exposed to ultrasound. That is, by the formation ofaggregates, there may be formed a pore space in and/or on the lipidbilayer of the liposomes.

The sonosensitizer may have Formula (I) below:<Formula (I)>A-B-C  (I)

In Formula (I), A is may be a moiety including an aromatic ring, B maybe a moiety including hydrogen bond donor and acceptor, and C may be ahydrophobic moiety including 8 to 40 carbon atoms.

In Formula (I), A may be an unsubstituted or substituted C₆-C₃₀ arylgroup or an unsubstituted or substituted C₃-C₃₀ heteroaryl group. A maybe, for example, a polycyclic aromatic hydrocarbon, and moreparticularly, a polycyclic aromatic hydrocarbon including at least twoor three rings. A may induce π-π stacking interactions between thesonosensitizers, and accordingly, the self-assembly of thesonosensitizers may be induced. The self-assembly may also enhance thepermeability of the liposomes. A may be a group having carbon numbers C3to C30, C3 to C20, C3 to C15, C3 to C10, C4 to C30, C4 to C20, C4 toC15, C4 to C10, C5 to C30, C5 to C20, C5 to C15, C5 to C10, C6 to C30,C6 to C25, C6 to C20, C6 to C15, or C6 to C12.

The term “aryl” as used herein is used alone or in combination, andrefers to an aromatic hydrocarbon group having one or more rings.

The term “aryl” also refers to a group in which an aromatic ring isfused to one or more cycloalkyl rings.

Examples of “aryl” include phenyl, naphthyl, and anthracene. At leastone hydrogen atom of the aryl group may be substituted with a halogenatom, a C₁-C₂₀ alkyl group substituted with a halogen atom (e.g., CCF₃,CHCF₂, CH₂F, and CCl₃), a C₁-C₂₀ alkoxy group, a C₂-C₂₀ alkoxyalkylgroup, a hydroxy group, a nitro group, a cyano group, an amino group, anamidino group, a hydrazine group, a hydrazone group, a carboxyl group ora salt thereof, a sulfonyl group, a sulfamoyl, a sulfonic acid group ora salt thereof, a phosphoric acid or a salt thereof, a C₁-C₂₀ alkylgroup, a C₂-C₂₀ alkenyl group, a C₂-C₂₀ alkynyl group, a C₁-C₂₀heteroalkyl group, a C₆-C₂₀ aryl group, a C₆-C₂₀ arylalkyl group, aC₆-C₂₀ heteroaryl group, a C₇-C₂₀ heteroarylalkyl group, a C₆-C₂₀heteroaryloxy group, a C₆-C₂₀ heteroaryloxyalkyl group, or a C₆-C₂₀heteroarylalkyl group. The substituents may have 1 to 20 carbon atoms,for example, 1 to 15 carbon atoms, 1 to 10 carbon atoms, 1 to 10 carbonatoms, 1 to 5 carbon atoms, 2 to 20 carbon atoms, 2 to 15 carbon atoms,2 to 10 carbon atoms, 2 to 5 carbon atoms, 3 to 20 carbon atoms, 3 to 15carbon atoms, 3 to 10 carbon atoms, 3 to 5 carbon atoms, 5 to 20 carbonatoms, 5 to 15 carbon atoms, 5 to 10 carbon atoms, 6 to 20 carbon atoms,6 to 15 carbon atoms, or 6 to 10 carbon atoms.

The term “heteroaryl” as used herein refers to a monocyclic or bicyclicorganic group that contains at least one ring where one or more heteroatoms selected from N, O, P, and S are ring atoms, and the remainingring atoms are carbon atoms. The heteroaryl group may include, forexample, 1 to 5 hetero atoms, and 5 to 10 ring members.

S or N may be oxidized to various oxidation states.

Typical monocyclic heteroaryl groups may include thienyl, furyl,pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, isothiazol-3-yl,isothiazol-4-yl, isothiazol-5-yl, oxazol-2-yl, oxazol-4-yl, oxazol-5-yl,isooxazol-3-yl, isooxazol-4-yl, isooxazol-5-yl, 1,2,4-triazol-3-yl,1,2,4-triazol-5-yl, 1,2,3-triazol-4-yl, 1,2,3-triazol-5-yl, tetrazolyl,pyrid-2-yl, pyrid-3-yl, 2-pyrazin-2-yl, pyrazin-4-yl, pyrazin-5-yl,2-pyrimidin-2-yl, 4-pyrimidin-2-yl, or 5-pyrimidin-2-yl.

The term “heteroaryl” also refers to a group in which a heteroaromaticring is fused to one or more aryl, cyclyaliphatic, or heterocyclicrings.

Examples of bicyclic heteroaryl include purinyl, indolyl, isoindolyl,indazolyl, indolizinyl, purinyl, quinolizinyl, quinolinyl,isoquinolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, quinazolinyl,quinaxalinyl, phenanthridinyl, phenathrolinyl, phenazinyl,phenothiazinyl, phenoxazinyl, benzisoqinolinyl, thieno[2,3-b]furanyl,furo[3,2-b]-pyranyl, 5H-pyrido[2,3-d]-o-oxazinyl,1H-pyrazolo[4,3-d]-oxazolyl, 4H-imidazo[4,5-d]thiazolyl,pyrazino[2,3-d]pyridazinyl, imidazo[2,1-b]thiazolyl,imidazo[1,2-b][1,2,4]triazinyl, 7-benzo[b]thienyl, benzoxazolyl,benzimidazolyl, benzothiazolyl, benzoxapinyl, benzoxazinyl,1H-pyrrolo[1,2-b][2]benzazapinyl, benzofuryl, benzothiophenyl,benzotriazolyl, pyrrolo[2,3-b]pyridinyl, pyrrolo[3,2-c]pyridinyl,pyrrolo[3,2-b]pyridinyl, imidazo[4,5-b]pyridinyl,imidazo[4,5-c]pyridinyl, pyrazolo[4,3-d]pyridinyl,pyrazolo[4,3-c]pyridinyl, pyrazolo[3,4-c]pyridinyl,pyrazolo[3,4-d]pyridinyl, pyrazolo[3,4-b]pyridinyl,imidazo[1,2-a]pyridinyl, pyrazolo[1,5-a]pyridinyl,pyrrolo[1,2-b]pyridazinyl, imidazo[1,2-c]pyrimidinyl,pyrido[3,2-d]pyrimidinyl, pyrido[4,3-d]pyrimidinyl,pyrido[3,4-d]pyrimidinyl, pyrido[2,3-d]pyrimidinyl,pyrido[2,3-b]pyrazinyl, pyrido[3,4-b]pyrazinyl,pyrimido[5,4-d]pyrimidinyl, pyrazino[2,3-b]pyrazinyl, orpyrimido[4,5-d]pyrimidinyl.

At least one hydrogen atom of the “heteroaryl” group may be substitutedwith the same substituents as described above in connection with thearyl group.

In Formula (I), A may be, for example,

wherein D₁ may be

in which at least one hydrogen atom of D1 may be substituted the samesubstituent as described above in connection with the aryl group. InFormula (I), A may be also

In Formula (I), B may be —NH—C(O)—NH—, —O—C(O)—, —NH—C(O)—, —CH(COOH)—,—NH—C(O)—O—, —S—, or a C₁-C₂₀ aliphatic hydrocarbon including at leastone substituent selected from the group consisting of —NH—C(O)—NH—,—O—C(O)—, —NH—C(O)—, —CH(COOH)— —NH—C(O)—O—, and —S—. The C₁-C₂₀aliphatic hydrocarbon may be a substituted or unsubstituted C₁-C₂₀ alkylgroup, a substituted or unsubstituted C₂-C₂₀ alkenyl group, or asubstituted or unsubstituted C₂-C₂₀ alkynyl group. At least one hydrogenatom of the “C₁-C₂₀ aliphatic hydrocarbon” may be substituted with thesame substituents as described above in connection with the aryl group.In Formula (I), B may include, for example, at least one selected fromthe group consisting of —NH—C(O)—NH—, —(CH₂)—O—C(O)CH₂CH(COOH)—,—S(CH₂)CH₂NHC(O)NH—, —(CH₂)₂NHC(O)(CH₂)₃NHC(O)O—, and —(CH₂)₆NHC(O)O—.

In Formula (I), C may be a hydrophobic moiety including 8 to 40 carbonatoms. The hydrophobic moiety including 8 to 40 carbon atoms may beselected from a substituted or unsubstituted C₁-C₄₀ alkyl group, asubstituted or unsubstituted C₁-C₄₀ alkoxy group, a substituted orunsubstituted C₂-C₄₀ alkenyl group, a substituted or unsubstitutedC₂-C₄₀ alkynyl group, a substituted or unsubstituted C₄-C₄₀ carbocyclicgroup, a substituted or unsubstituted C₅-C₄₀ carbocyclicalkyl group, asubstituted or unsubstituted C₄-C₄₀ carbocyclic oxy group, and asubstituted or unsubstituted C₅-C₄₀ carbocyclic alkyloxy group. At leastone hydrogen atom of the hydrophobic moiety including 8 to 40 carbonatoms may be substituted with the same substituents described above inconnection with the aryl group. In Formula (I), C may be a group havingcarbon numbers C8-C40, C8-C30, C8-C26, C8-C20, C8-C18, C8-C14, C10-C40,C10-C30, C10-C26, C10-C20, C10-C18, C10-C14, C12-C40, C12-C30, C12-C26,C12-C20, C12-C18, C12-C14, C14-C40, C14-C30, C14-C26, C14-C20, orC14-C18. In Formula (I), C may be a C₈-C₄₀ alkyl group, for example, analkyl group such as straight alkyl having carbon numbers C8, C10, C12,C14, C16, C18, C20, C22, C24, or C26. In addition, in Formula (I), C maybe a sterol or a derivative thereof. The sterol or the derivativethereof may be cholesterol or a derivative thereof, or squalene or aderivative thereof.

The sonosensitizer may include, for example, a structural formula of

wherein C₁ may be a C₈-C₄₀ alkyl group, for example, an alkyl group suchas straight alkyl having carbon numbers C8, C10, C12, C14, C16, C18,C20, C22, C24, or C26, B₁ may be —NH—C(O)—NH—, —O—C(O)—, —NH—C(O)—,—CH(COOH)—, or —NH—C(O)—O—, and D₁ is the same as described above. Thesonosensitizer may have a structural formula of

The sonosensitizer, in a state not exposed to ultrasound, may becontained in the liposome in a concentration less than a criticalconcentration at which the structure of the liposome may be destroyed.The sonosensitizer may be contained in the liposome in a range of fromabout 0.1% to about 20%, for example, about 0.1% to about 5%, about 1%to about 20%, about 2% to about 20%, about 4% to about 20%, about 6% toabout 20%, about 8% to about 20%, about 10% to about 20%, about 1% toabout 18%, about 1% to about 16%, about 1% to about 14%, about 1% toabout 12%, about 1% to about 10%, about 1% to about 5%, about 2% toabout 18%, about 2% to about 16%, about 2% to about 14%, about 2% toabout 12%, about 2% to about 10%, or about 2% to about 5%, based on thetotal weight of the lipid molecules.

The sonosensitizer may not be a polymer including ethylene glycol. Thepolymer including ethylene glycol may be Triton™ X-100, Triton™ X-405,Tween™ 20, Tween™ 80, polyethylene glycol (PEG), polypropylene (PPO), aPEG triblock copolymer such as Pluronic P-105, or any combinationthereof.

The liposome may further include an active agent. The active agent maybe a pharmaceutical active agent, a magnetic active agent, an imagingagent, or any combination thereof. The active agent may be, for example,a compound, a protein, a peptide, nucleic acid, a nanoparticle, or anycombination thereof. The active agent may include, for example, ananticancer agent, an anti-angiogenesis inhibitor, an anti-inflammatoryagent, an analgesic, an antiarthritic, a dedative, an antidepressant, anantipsychotic agent, a tranquilizer, a tranquilizer, an antianxietyagent, a narcotic antagonist, an antiparkinson agent, a cholinergicagonist, an immunosuppressive agent, antiviral agent, an antibiotic, anappetite suppressant, an anticholinergic agent, an antihistamine, ananti-migraine agent, a hormone, a vasodilator, a contraceptive, anantithrombotic, a diuretic, an antihypertensive drug, a cardiovasculartherapeutic, a wrinkle-diminishing agent, a skin aging inhibitor, askin-whitening agent, or any combination thereof.

The active agent may be a hydrophobic drug, and examples thereof includesorafenib, paclitaxel, cyclosporine A, amphothericin B, indinavir, orany combination thereof. Sorafenib may be used as a therapeutic agentfor renal cancer and liver cancer. Paclitaxel may be used as atherapeutic agent for ovarian cancer, breast cancer, or lung cancer.Cyclosporine A may be used as an immunosuppressive drug. Amphothericin Bmay be used as a polyene antibiotic. Indinavir may be used as a proteaseinhibitor. The hydrophobic active agent may be a steroid-based material,and examples thereof include glucocorticoid, a taxane-based drug, acyclic peptide-based drug (e.g., cyclosporine A), indinavir,amphotericin B, or any combination thereof. Hydrophobic glucocorticoidmay include, for example dexamethasone, trimacinolone, beclomethasonediproprionate, triamcinolone acetate, diacetate, bethamethasonediproprionate, testosterone, budesonide, 17α-ethinylestradiol,levonorgestrel, fluticasone proprionate, or any combination thereof. Theactive agent may be also a hydrophilic active agent. The term“hydrophilic” as used herein indicates properties of easy binding towater molecules, water solubility, or the nature of polarity. Forexample, the hydrophilic active agent may include methotrexate,doxorubicin, epirubicin, daunorubicin, vincristine, vinblastine,etoposide, ellipticine, camptothecin, docetaxel, cisplatin, prednisone,methyl-prednisone, biprobufen, idarubicine, valrubicin, mitoxantrone,ampicillin, streptomycin, penicillin, or any combination thereof.

The active agent may be a peptide drug, a protein drug such as anantibody, a biomolecule other than the peptide drug and the proteindrug, or any combination thereof. Also, the active agent may be acombination of a chemical drug. The active agent may be, for example,Avastin® (Genentech/Roche), or a combination of Avastin® withchemotherapeutic agents, such as 5-fluorouracil, leucovorin,oxaliplatin, and irinotecan.

The term “imaging agent” as used herein is used interchangeably with theterm “contrast media”. The imaging agent refers to a material to enhancecontrast of an image that shows tissues or blood vessels clearly at thetime of examination such as magnetic resonance imaging (MRI) or computedtomography (CT) by artificially increasing absorption differences ofeach tissue or blood vessel. The imaging agent may be classified into anegative imaging agent and a positive imaging agent. The negativeimaging agent allows more penetration than other surrounding tissues do,so as to show an image. For example, the positive imaging agent may bean iodine-containing imaging agent or barium sulfate, and the negativeimaging agent may be air, gas, or carbon dioxide. The imaging agent maybe a transitional element or a chelate complex of the transitionalelement. The transitional element may be, for example, La, Pr, Nd, Gd,Tb, Mn, Zn, Fe, Sc, Ti, V, Zn, Y, Zr, Nb, Mo, Pd, Ag, Cd, W, or Re. Thetransitional element may be in the form of ions. For example, gadolidium(atomic symbol Gd and atomic number 64) may be in the form of Gd³⁺. Achelate complex of Gd may include, for example, gadoteric acid,gadodiamide, gadobenic acid, gadopentetetic acid, gadoteridol,gadoversetamide, gadoxetatic acid, gadobutrol, or any combinationthereof.

The active agent may be contained in an interior space of the liposome,in an interior of the lipid bilayer, or in both.

The liposome may have a diameter of 20 nm or greater, for example, in arange from about 50 nm to about 500 nm, about 50 nm to about 400 nm,about 50 nm to about 300 nm, about 50 nm to about 200 nm, or about 50 nmto about 150 nm. The liposome may be in a form of unilamellar vesicles(SUVs) or multivesicular vesicles.

The liposome may be manufactured according to methods widely known inthe art. The liposome may be manufactured by a method using a thin filmhydration technique. The liposome may use an aqueous solution thereof asa hydrating fluid of water-soluble (hydrophilic) materials, or may add adrug or a drug solution at any step in the manufacturing of theliposome, thereby manufacturing a liposome in which the water-solublematerials are entrapped. In addition, fat-soluble (hydrophobic)materials may be prepared by which the materials are dissolved in anorganic solution of a configuration lipid, and the mixed solution isevaporated to obtain a dried lipid film containing drugs and to behydrated. Such methods are related to a step of loading an active agent(i.e., a passive loading) before or during the manufacturing of theliposome. However, a specific type of compound, such as a compoundhaving an ionizable group or a material having both lipid and watersolubility, may be introduced into the liposome (i.e., a remote loading)after these specific type of compounds are formed as intact vesicles. Anexample of the remote loading includes an ammonium sulfate gradientmethod (J. Control. Release 2009, 139, 73-80).

According to another aspect of the present disclosure, a pharmaceuticalcomposition for delivering an active agent to a subject includes aliposome including a lipid bilayer, a sonosensitizer disposed on thelipid bilayer, and an active agent, wherein the sonosensitizerself-assembles to form aggregates, when exposed to ultrasound.

A detailed description of the sonosensitive liposome including the lipidbilayer and the sonosensitizer disposed on the lipid bilayer, whereinthe sonosensitizer self-assembles to form aggregates, when exposed toultrasound, has been already described above.

The pharmaceutical composition may further include a pharmaceuticallyacceptable carrier or diluent. The pharmaceutically acceptable carrieror diluent may be well known in the art, and examples thereof includelactose, dextrose, sucrose, sorbitol mannitol, starch, acacia gum,calcium phosphate, alginate, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water (e.g., saline orsterile water), syrup, methyl cellulose, methylhydroxybenzoate,propylhydroxybenzoate, talc, magnesium stearate, mineral oil, Ringer'ssolution, buffer, maltodextrin solution, glycerol, ethanol, or anycombination thereof. The pharmaceutical composition may further includea lubricant, a wetting agent, a sweetening agent, a flavoring agent, anemulsifier, a suspending agent, a preserving agent, or any combinationthereof.

According to methods that are known in the art, the pharmaceuticalcomposition may be formulated and prepared in the form of a unit doseusing the pharmaceutically acceptable carrier and/or diluents, or may beintroduced and prepared in a multi-dose container. Here, thepharmaceutical composition may be formulated in types of a solution ofoil or aqueous medium, suspension, syrup, or emulsion. In someembodiments, the pharmaceutical composition may be formulated in typesof extracts, powders, powdered drugs, granules, tablets, or capsules.The pharmaceutical composition may further include a dispersant or astabilizer. The aqueous medium may contain physiological saline or PBS.

According to another aspect of the present disclosure, a method ofdelivering an active agent into the body of a subject includesadministering a pharmaceutical composition including a liposomeincluding a lipid bilayer, a sonosensitizer disposed on the lipidbilayer, and an active agent, wherein the sonosensitizer self-assemblesto form aggregates, when exposed to ultrasound; and applying ultrasoundto the subject to release the active agent.

The method includes administering the pharmaceutical compositionincluding the liposome including the lipid bilayer, the sonosensitizerdisposed on the lipid bilayer, and the active agent, wherein thesonosensitizer self-assembles to form aggregates, when exposed toultrasound.

A detailed description of the liposome including the lipid bilayer, thesonosensitizer disposed on the lipid bilayer, and the active agent hasbeen already described above.

The subject may be a mammal, and the mammal may be a primate mammal. Thesubject may be a human, a cow, a pig, a horse, a rabbit, a mouse, or acombination thereof.

The administration may be, for example, oral administration orparenteral administration. The parenteral administration may be, forexample, intravenous, intramuscular, intracavity (abdominal cavity,joints, or eyes), or direct injection. The direct injection may involveinjecting directly into a diseased site such as a tumor site. Theliposome may be administered intravenously, and accordingly, brought tothe target site such as a tumor site by blood flow. The target site mayhave a leaky property. Dosage of the liposome may be prescribedaccording to various factors such as formulation methods, administrationmethods, patient's age, weight, gender and morbidity, foods,administration times, administration routes, excretion rates, andreaction sensitivity. Dosage of the liposome may be in a range fromabout 0.001 mg/kg to about 100 mg/kg, for example, about 0.01 mg/kg toabout 100 mg/kg, about 0.1 mg/kg to about 100 mg/kg, about 1 mg/kg toabout 100 mg/kg, about 10 mg/kg to about 100 mg/kg, about 50 mg/kg toabout 100 mg/kg, about 0.001 mg/kg to about 50 mg/kg, about 0.01 mg/kgto about 10 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 1 mg/kg toabout 10 mg/kg, or about 1 mg/kg to about 50 mg/kg.

The administration may be carried out in various ways including use of acatheter. The catheter is a device that may be inserted into a subject,and more particularly, a tubular structure like blood vessels, digestivetracts, ureters, and reproductive organs. A typical structure of thecatheter is known in the art. The catheter is typically flexible and hasa tubular main body including one or more lumens extending through thetubular main body. In order to inject liposome-like vesicles into asubject, there may be a port placed at a terminus of the catheter.Attachment of an ultrasound imaging probe to the catheter may be helpfulin visualizing an inner wall of the tubular structure of the subject, todeliver the liposomes containing active agents such as a therapeuticagent or a diagnostic agent to a guided and targeted specific area ofthe subject. A material that specifically binds to a target cell may be,for example, an antibody that specifically binds to an antigen presenton the surface of the target cell, or a liposome to which anantigen-binding fragment thereof is bound, and such a material mayspecifically bind to a target cell. In addition, a material thatspecifically binds to a substance in the body fluid may be, for example,an antibody that specifically binds to an antigen of the body fluid, ora liposome to which an antigen-binding fragment thereof is bound, andsuch a material may be specifically moved to a specific body fluid.

The administration may be site-specific administration in the subject.The specific site may include tumor or brain.

The method includes applying ultrasound to the target site of thesubject to release the active agent.

Ultrasound may be irradiated at amplitudes of 5% to 100% or 20% to 100%of the maximum amplitude. Ultrasound may be irradiated so as to have anoutput range from about 10 W/cm² to about 15 kW/cm².

Ultrasound may be applied to the subject for about 1 to about 20minutes, for example, about 5 to about 15 minutes. The ultrasound may berandomly applied to the entire subject, or applied to a specific site ofthe subject.

One or more embodiments of the present disclosure will now be describedmore fully with reference to the following examples. However, theseexamples are provided only for illustrative purposes and are notintended to limit the scope of the present disclosure.

Example 1 Sonosensitive Liposome Size and Drug Release, According toTypes of Phospholipids

Liposomes including sonosensitizers were prepared. Then, it wasconfirmed that sizes of the liposomes were changed according to theamount of the sonosensitizers, and sizes of the liposomes and drugrelease of the liposomes were changed when exposed to ultrasound. A2′-deoxyadenosine derivative having a urea linker and an octylhydrocarbon tail of Formula 2 below, was used as the sonosensitizer. The2′-deoxyadenosine derivative was synthesized according to methodsdescribed in the literature (Soft matter, 2008, 4, 1995-1997).

Doxorubicin (DX) was loaded on the liposomes according to the ammoniumsulfate gradient method (J. Control. Release 2009, 139, 73-80).

In greater detail, liposomes in a form of unilamellar vesicles wereprepared using a lipid thin film hydration method as follows. First,1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) (DSPE-PEG), and cholesterol were obtainedfrom Avanti Polar Lipid company (USA)). A 2′-deoxyadenosine derivativehaving an octyl hydrocarbon tail including a urea moiety of Formulaabove was used as the sonosensitizer. The 2′-deoxyadenosine derivativewas synthesized according to methods described in the literature (Softmatter, 2008, 4, 1995-1997). DPPC/DSPC, DSPE-PEG 2000, cholesterols, andsonosensitizer were prepared in a molar ratio of 55:2:15:0.55, anddissolved in chloroform in a round-bottom flask. The total lipidconcentration was about 9.6 mg/ml. A lipid thin layer was formed on theinterior wall of the flask by evaporating chloroform at room temperatureunder reduced pressure using a rotary evaporator.

Next, the lipid thin layer was hydrated by adding a 250 mM ammoniumsulfate solution (pH 4.0) to the flask, and the hydrated solution wassubjected to vortexing. Unilamellar vesicle type liposomes were preparedby filtering the hydrated solution at room temperature through apolycarbonate film with pores having a size of 100 nm.

A liposome solution formed of liposomes with 250 nM of ammonium sulfateinside and 25 mM of Tris.HCl outside by passing the prepared unilamellarvesicle type liposomes solution through a PD-10 desalting column (GEHealthcare) including Sephadex™ G-25 medium filled with 25 nM ofTris.HCl (pH 9.0) (Sample 1). 100 ul (10 mg/ml) of doxorubicin (DX) wasadded to the obtained liposome solution, and then, incubated for an hourat a temperature of 37° C. (in a mass ratio of 1:0.2 to the main lipidcomponents). The prepared liposome solution was passed through a PD-10Desalting Column (GE Healthcare) including Sephadex™ G-25 medium filledwith PBS buffer to remove unsealed DX. As a result, liposomes in whichDX was entrapped in the aqueous interior were prepared (Sample 2). Thesizes of the liposome particles in the prepared Samples 1 and 2 weremeasured by a Zeta-sizer device (Malvern inst.) using dynamic lightscattering (DLS). The results of the sizes of the liposome particleswere shown in Table 1 below.

TABLE 1 Sample 1 Sample 2 Primary lipid d (nm) Pdi d (nm) Pdi DPPC 166.60.0475 169.3 0.087 DSPC 183.3 0.1305 186.55 0.111

Referring to Table 1, Samples 1 and 2 each showed the liposomes withunsealed DX and liposomes with sealed DX, and d and Pdi each indicated adiameter and a number polydispersity index of the liposome particles. Asshown in Table 1, the sizes of the liposomes were different according tothe types of the primary lipids, and the injection of DX did not inducesignificant changes in the particle sizes.

Example 2 Changes in Liposome Sizes According to Exposure Time toUltrasound

Liposomes were prepared according to the same method used in Example 1,except that DPPC, DSPE-PEG, cholesterols and sonosensitizers wereprepared in a molar ratio of 55:2:15:0.55, 55:2:15:1.1, or 55:2:15:2.75.In this case, senosensitizers contents in mol % with respect to DPPC is1%, 2% and 5%, respectively.

Then, the sizes of the prepared liposomes were measured according to thesame method used in Example 1. In addition, dispersions of the preparedliposomes were diluted using a PBS buffer in a ratio of 1:10 by volume,and 5 ml of the diluted solution was put in a sample chamber of a VibraCell ultrasonic processor (VCX 130, Sonics & Materials Inc.). Here,ultrasound having a frequency of 20 kHz and an output of 130 W wasapplied thereto for a specified period of time. Ultrasound was appliedthereto at amplitudes of 100% to 50% of the maximum amplitude. Tables 2and 3 each showed changes in the sizes of the liposomes according to theamounts of sonosensitizers (Table 2) and the exposure time to ultrasound(Table 3).

TABLE 2 Amounts of sonosensitizer Sample 1 Sample 2 (mol %) d (nm) Pdi d(nm) Pdi 1 174.8 0.0475 169.3 0.087 2 170.15 0.0355 171.15 0.085 5166.45 0.0575 165.15 0.0945

Referring to Table 2, Samples 1 and 2 each showed liposomes withunsealed DX and liposomes with sealed DX, and d and pdi each indicated adiameter and a number polydispersity index of the particles. In Table 2,the amounts of the sonosensitizers were indicated in mol % with respectto the main lipid DPPC. As shown in Table 2, as the amounts of thesonosensitizers increased, the liposome particle sizes were found to besmaller. Data in Table 2 were obtained by measuring the sizes of theliposomes prepared according to the amounts of the sonosensitizers, andthe sizes of the liposomes with unsealed DX without application ofultrasound.

TABLE 3 Ultrasound exposure time Sample 1 (minutes) d (nm) Pdi 1 164.60.1105 2 164.2 0.117 5 164.9 0.074 10 165.05 0.062

Referring to Table 3, Sample 1 showed the liposomes with unsealed DX,and d and Pdi each indicated a diameter and a number polydispersityindex of the particles. As shown in Table 3, it was confirmed that thesizes of the liposomes were not changed according to the exposure timeto ultrasound. As a result, it was confirmed that the liposomes did notdestroy themselves in spite of the exposure to ultrasound.

It was also confirmed that the drug release from the liposomes withsealed DX was changed (increased) according to the exposure time toultrasound (see FIG. 1). As a result, it was confirmed that the drugrelease may be induced by the exposure to ultrasound.

Example 3 Changes in Drug Release of Liposomes According to Molar Ratiosof Sonosensitive Molecules and Stimulus Applied Thereto

Liposomes were prepared according to the same method used in Example 1,except that DPPC, DSPE-PEG, cholesterols and sonosensitizers wereprepared in a molar ratio of 55:2:15:0.275, 55:2:15:0.55, 55:2:15:1.1,55:2:15:1.65, 55:2:15:2.2, or 55:2:15:2.75.

The drug release of the prepared liposomes were measured by applyingultrasound (having a frequency of 20 kHz and an output of 130 W)thereto, according to the same method used in Example 2, and measuringthe extent of the drug release according to DX fluorescence using afluorescence spectrometer (PerkinElmer, Envision 2104-multilabelreader). The measured fluorescence was indicated as a percentage withrespect to the total amount of DX. In greater detail, after ultrasoundwas applied to the liposomes, the fluorescent intensity of the samplewas appropriately diluted to determine the amounts of DX released fromthe liposomes, and then measured at an excitation wavelength (λex) of485 nm and an emission wavelength (λem) of 635 nm. The relativepercentage in fluorescence intensity (% release) according to theapplication of ultrasound during a specific period of time wascalculated based on the total release of the entrapped substances thatwere obtained after the destruction of the liposomes by adding 1% TritonX-100 (ethanol). Percent (%) release was calculated according toEquation below.% release=(Ft−Fi)/(Ff−Fi)×100

(*Ft is a fluorescence value measured according to the ultrasoundexposure time; Fi is a fluorescence value measured before the ultrasoundexposure; and Ff is 1% Triton X in EtOH)

In addition, in order to confirm the thermal stability of the sameliposomes, the prepared liposomes were incubated for one hour at atemperature of 37° C., and the amount of the DX released therefrom weremeasured as described above.

FIG. 1 is a graph showing the extent of drug release according toamounts of the sonosensitizers and ultrasound exposure time. In FIG. 1,the amounts of the sonosensitizers were indicated in mol % with respectto the main lipid DPPC. As shown in FIG. 1, the extent of the drugrelease varies according to the amounts of the sonosensitizers, andthus, the extent of the drug release may be controlled by adjusting theamounts of the sonosensitizers.

When the liposomes containing DX including a 2 mol % sonosensitizer wereexposed to ultrasound for about 3 minutes, the drugs of about 90% ormore has been released. In FIG. 1, the US exposure time on thehorizontal axis indicates the exposure times to ultrasound and the Doxrelease on the vertical axis indicates the extents of the Dox release,and 0.5 mol %, 1 mol %, 2 mol %, 3 mol %, 4 mol %, and 5 mol % indicatethe amounts of the sonosensitizers.

Table 4 is the result of FIG. 1.

TABLE 4 Exposure time to Amounts of sonosensitizer ultrasound 0.5% 1% 2%3 4 5 (minute) DX release (%) 1 19.8 31.6 35.9 27.9 24.3 22.6 3 46.354.0 87.5 54.0 50.9 56.5 5 65.7 66.4 89.6 66.3 62.4 76.1 10 84.2 76.885.2 72.2 75.09 90.5

Table 5 shows the extent of the drug release measured when the liposomescontaining the sonosensitizers and DX were incubated for one hour at atemperature of 37° C.

TABLE 5 Amounts of sonosensitizer (mol %) Drug release (%) 0.5 0 10.7386 2 2.8601 3 1.3585 4 0.8075 5 0.5267

Referring to Table 5, the extent of the drug release was found to beless than about 2.9%, which means that the drug only barely released.Therefore, it was confirmed that the liposomes were not sensitive totemperature changes, and the permeability thereof was changed accordingto the exposure to ultrasound.

Example 4 Observation of Cellular Uptake of SonosensitiveLiposome-Treated Cells

Liposomes were prepared according to the same method used in Example 1,except that DPPC, DSPE-PEG, cholesterols and sonosensitizers wereprepared in a molar ratio of 55:2:20:1.65. The drug release of theprepared liposomes was induced by applying ultrasound (having afrequency of 20 kHz and an output of 130 W, at 50% amplitude) theretofor a given time 5 minutes, according to the same method used in Example2. Hela cells were grown in a 5% CO₂ incubator filled with DMEM (Gibco)supplemented with 10% bovine serum albumin (BSA) at a temperature of 37°C. 10%. All cells were subcultured twice a week. The cells were seededper well in concentration of 1×10⁶ cells in a 12-well plate. The cellsgrew for 24 hours, so as to have a confluence of about 80% before theexperiment. The cells were washed twice with a DMEM culture medium, andthen, 10 ul of the sonicated liposomes (DX concentration of 20 ug/ml)was immediately added to each well of the plate, and incubated for 30minutes under the same conditions. Next, the fluorescence intensitycoming from the cells were measured by a confocal microscope (LSm 710,Carl Zeiss, USA). As a result, the drugs released by the exposure toultrasound, and the cellular uptake of the released drugs wereconfirmed. When the ultrasound was applied at an amplitude of 50% of themaximum amplitude and at a frequency of 20 kHz to the liposomes, thedrug release and the cellular uptake were increased proportional to theexposure time to ultrasound. When the liposomes were exposed toultrasound for more than 1 minute, the extent of the drug release andthe cellular uptake were similarly shown.

The same liposomes were exposed to ultrasound for 5 minutes at differentamplitudes (i.e., 50%, 75%, and 100% of the maximum amplitude) at afrequency of 20 kHz. 10 ul of the sonicated liposomes (DX concentrationof 20 ug/ml) was immediately added to Hela cells as described above, andthen, incubated for 30 minutes. As a control group, a group treated with10 ul of PBS, a group treated with 10 ul of radical DX (20 ug/ml ofdistilled water), and a group wherein HeLa cells are incubated at atemperature of 42° C. were all included. The cell viability thereof wasobtained by counting viable cells using a WST assay[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salts] (WST-8, Cell Counting Kit-8, Dojindo, Japan).

FIG. 2 is a graph showing effects of liposome on cell viability, theliposomes containing drugs that are irradiated at various amplitudes. Asshown in FIG. 2, in the case of exposure to ultrasound for 5 minutes atan amplitude of 50% (Dox@sonoliposome (U50%, 5 min)), 75%(Dox@sonoliposome (U75%, 5 min)), and 100% (Dox@sonoliposome (U100%, 5min)) of the maximum amplitude, and at a frequency of 20 kHz, the cellviability of the liposomes was about 15% compared to that of the controlgroup (in which DX was not treated), and that is, the cell viability ofthe liposomes was similar with that of the liposomes in which radical Dxwas treated. Meanwhile, in a case that Hela cells were incubated at atemperature of 42° C. without ultrasound treatment or any othertreatment, the cell viability thereof was more than about 100. Thus, itwas confirmed that the liposomes did not destroy themselves at atemperature of 42° C., or the permeability thereof did not increase at atemperature of 42° C. In FIG. 2, the PBS indicates a group treated with10 ul of PBS only, the Dox (20 ug/ml) indicates a group treated with 10ul of radical DX (20 ug/ml of distilled water), the Dox@sonoliposome (T)indicates a group treated with HeLa cells at a temperature of 42° C.,the Dox@sonoliposome (U50%, 5 min), the Dox@sonoliposome (U75%, 5 min),and the Dox@sonoliposome (U100%, 5 min) each indicates ultrasoundexposure for 5 minutes at an amplitude of 50%, 75%, and 100% of themaximum amplitude and at a frequency of 20 kHz.

As described above, the liposomes including the sonosensitizers do notdestroy themselves at temperature changes nor increase the permeabilitythereof. However, the liposomes increase the permeability thereof inresponse to ultrasound. In this regard, the liposomes may deliver drugsto a subject according to ultrasound-specific ways.

As described above, according to the one or more of the aboveembodiments of the present disclosure, a sonosensitive liposome and apharmaceutical composition including the same may be used to efficientlydeliver the liposome or an active agent contained therein. According toanother or more of the above embodiments of the present disclosure, amethod of delivering the active agent to a target site in the body of asubject may be used to efficiently deliver the active agent to thetarget site in the body of the subject.

It should be understood that the exemplary embodiments described thereinshould be considered in a descriptive sense only and not for purposes oflimitation. Descriptions of features or aspects within each embodimentshould typically be considered as available for other similar featuresor aspects in other embodiments. While one or more embodiments of thepresent disclosure have been described with reference to the figures, itwill be understood by those of ordinary skill in the art that variouschanges in form and details may be made therein without departing fromthe spirit and scope of the present disclosure as defined by thefollowing claims.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. A method of delivering an active agent to asubject, the method comprising: administering a liposome comprising alipid bilayer, a sonosensitizer, and an active agent to a subject; andapplying ultrasound to the liposome to release the active agent; whereinthe sonosensitizer is disposed in and/or on the lipid bilayer; whereinthe sonosensitizer has the structural formula

wherein C₁ is a C₈-C₄₀ alkyl group, B₁ is —NH—C(O)—NH—, —O—C(O)—,—NH—C(O)—, —CH(COOH)—, or —NH—C(O)—O—, and D₁ is selected from the groupconsisting of:

and wherein the sonosensitizer self-assembles to form aggregates whenexposed to ultrasound.
 2. The method of claim 1, wherein thesonosensitizer has a structural formula of


3. The method of claim 1, wherein the active agent is a pharmaceuticalactive agent, a magnetic active agent, an imaging agent, or anycombination thereof.
 4. The method of claim 3, wherein the active agentis contained in an interior space of the liposome, in an interior of thelipid bilayer, or in both.
 5. The method of claim 1, wherein thepermeability of the liposome increases when the liposome is exposed toultrasound.
 6. The method of claim 1, wherein exposure of the liposometo ultrasound causes the formation of a pore space in and/or on thelipid bilayer.
 7. The method of claim 1, wherein the lipid bilayercomprises a lipid molecule selected from the group consisting of aphospholipid, a lipid conjugated to polyethylene glycol (PEG),cholesterol, a cholesterol derivative, a steroid, or combinationsthereof.
 8. The method of claim 7, wherein the phospholipid is selectedfrom the group consisting of a phosphatidylcholine, a phosphatidic acid,a phosphatidylethanolamine, a phosphatidylserine, aphosphatidylinositol, a phosphosphingolipid, or combinations thereof. 9.The method of claim 8, wherein the phosphatidylcholine is selected fromthe group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), eggphosphatidylcholine, soy bean phosphatidylcholine, or combinationsthereof.
 10. The method of claim 1, wherein the lipid bilayer comprisesDPPC and egg phosphatidylcholine at a ratio ranging from about 4:1 toabout 1:4.
 11. The method of claim 10, wherein the lipid bilayercomprises DPPC and egg phosphatidylcholine at a ratio ranging from about3:1 to about 1:3.
 12. The method of claim 11, wherein the lipid bilayercomprises DPPC and egg phosphatidylcholine at a ratio ranging from about2:1 to about 1:2.
 13. The method of claim 12, wherein the lipid bilayercomprises DPPC and egg phosphatidylcholine at a ratio of 1:1.
 14. Themethod of claim 7, wherein the lipid conjugated to PEG is1,2-distearoylphosphatidylethanolamine-methyl-polyethylene glycol(DSPE-PEG).
 15. The method of claim 7, wherein the steroid is selectedfrom the group consisting of sitosterol, ergosterol, stigmasterol,4,22-stigmastadiene-3-on, stigmasterol acetate, lanosterol,cycloartenol, or combinations thereof.
 16. The method of claim 7,wherein the cholesterol, cholesterol derivative, or steroid enhancesstability of the lipid bilayer and assists to lower the permeability ofthe lipid bilayer.
 17. The method of claim 1, wherein the thickness ofthe lipid bilayer is from about 1 nm to about 10 nm.
 18. The method ofclaim 1, wherein the liposome has a diameter from about 50 nm to about500 nm.